Molecular Identification of Mycobacterium avium subsp. Paratuberculosis isolated from ELISA-Positive Samples by Nested PCR

Document Type : Research Article

Authors

• Mahsa Soleimani ¹
• Alireza Shahrjerdi ²
• Mitra Salehi ¹

¹ Department of Microbiology, Faculty of Biological Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran.

² National Institute for Genetic Engineering and Biotechnology, Tehran, Iran.

10.22067/ijvst.2023.83414.1280

Abstract

Paratuberculosis (Johne's disease) is a chronic granulomatous small intestine disease caused by MAP. Diagnosing and isolating infected animals is the most important measure for controlling the disease. Therefore, this study aimed to molecularly identify mycobacterium isolated from ELISA-positive cows with Johne's disease by nested PCR from the samples from Markazi Province, Iran. For this purpose, 2938 samples were decontaminated and then cultured on the Herrold egg culture medium containing mycobactin and no mycobactin. After DNA extraction, PCR for 16S rRNA was first performed, followed by nested PCR on positive samples. Of 2938 samples, 87 were positive, and 26 were suspected. All positive isolates were observed in Ziehl-Neelsen staining in microscopic expansion. A 543-bp band was observed in 26 tested samples and mycobacterium strains in PCR for 16S rRNA, indicating the presence of mycobacterium in the above samples. Nested PCR was performed for all isolates and positive and negative control strains. A 398-bp band was obtained in the first stage, and a 298-bp fragment was obtained in the second stage, indicating the presence of MAP in the samples. Accordingly, nested PCR is suggested as a proper method for the quick and definitive diagnosis of disease cases.

Keywords

• Mycobacterium avium
• Johne's disease
• 16S rRNA
• Nested PCR

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